Enhancing long-segmental tracheal restoration: A self-repairing hydrogel loaded with chondrocytokines for sutureless anastomosis and cartilage regeneration

Abstract

Artificial tracheal substitutes encounter significant challenges during long-segmental tracheal defects (LSTD) reconstruction, notably early postoperative anastomotic stenosis and tracheal chondromalacia. Mitigating early anastomotic stenosis by creating a compliant sutureless substitute is pivotal. Enhancing its chondrogenic capacity is equally critical for sustained healthy tracheal cartilage regeneration. This study proposes a self-healing hydrogel for sutureless tracheal anastomosis to mitigate anastomotic stenosis, enriched with kartogenin (KGN) and transforming growth factor-β1 (TGFβ1) to bolster chondrogenic properties. Initially, two precursor solutions were prepared: 1) aldehyde-modified hyaluronic acid with sulfonation and β-cyclodextrin-CHO loaded with KGN; 2) hydrazide-grafted gelatin loaded with TGFβ1. Coextrusion of these solutions resulted in a gelated G + TGFβ1/sH-CD + KGN hydrogel, characterized by a robust covalent bonding network of acylhydrazones between hydrazide and aldehyde groups, imparting excellent self-healing properties. The G + TGFβ1/sH-CD + KGN hydrogels, showcasing favorable cytocompatibility, excellent injectability, and rapid gelation, were loaded with bone marrow stem cells. These were customized into O-shaped rings and assembled into a malleable tracheal substitute using our established ring-to-tube method. This resultant compliant substitute facilitated sutureless anastomosis of LSTD in a rabbit model, attributed to the Schiff base reaction between the hydrogel's carbonyl group and the tissue's amino group. Notably, the tracheal substitute reduced early postoperative anastomotic stenosis, maintained tracheal patency, alleviated sputum blockage, promoted reepithelization, and increased the survival rate of the experimental rabbits. The sustained release of chondrocytokines resulted in excellent tracheal cartilage regeneration. Employing chondrocytokines-loaded hydrogels with self-healing properties represents a significant advancement in sutureless tracheal anastomosis and tracheal cartilage regeneration, holding promising potential in inhibiting early postoperative anastomotic stenosis and tracheal chondromalacia when treating LSTD.

Keywords: Dual-drug encapsulation; Hydrogel; Self-healing; Suture-free; Tracheal reconstruction.

Graphical abstract

Scheme 1

Development and application of G + TGFβ1/sH-CD + KGN. (A) Initially, two solutions were prepared: one composed of HA-CHO-SO₃ and β-CD-CHO loaded with KGN, while the other contained gelatin-ADH loaded with TGFβ1. These solutions were coextruded through a double-barreled syringe, forming ring-shaped hydrogels that were injected into O-shaped molds. The resulting gelated G + TGFβ1/sH-CD + KGN hydrogel demonstrated excellent self-healing properties due to the network of acylhydrazones formed between the hydrazide and aldehyde groups in the side chain. KGN was loaded onto β-CD-CHO and linked to the gelatin skeleton through a condensation reaction. TGFβ1 was captured through electrostatic interactions with the -SO3 group introduced in the HA skeleton. (B) Leveraging the self-healing characteristics, both BMSCs loaded and unloaded hydrogels in O-shape were arranged alternately using our previously established ring-to-tube method to create a tracheal substitute. The G + TGFβ1/sH-CD + KGN hydrogel exhibited excellent tissue adhesive properties due to the Schiff base reaction between the hydrogel's carbonyl group and the native tissue's amino group. These tracheal substitutes were utilized for sutureless anastomosis to repair LSTD in a rabbit model.

Fig. 1

Rheological analysis of the G/H hydrogels. Rheological time sweep test of (a) GxH₂ and (b) GxH₄ hydrogels, in which x marks 8, 10, 12, 14, 16, and 18. The solid symbols represented G′ and the hollow symbols represented G''. (c) Rheological cyclic low-high strain of G₁₈/H₄ hydrogel. (d) Rheological strain sweep test of G₁₈/H₄ hydrogel at 37 °C atmospheres.

Fig. 2

Injectability, self-healing and adhesive properties of the G/H hydrogels. (a) The injectable behavior demo of G/H hydrogel. (b) The self-healing process of G/H hydrogel in different models. (c) The adhesive tests of G/H hydrogel to G/H hydrogel (left panel) and G/H hydrogel to rabbit tracheal tissue (right panel). (d) SEM image of the freeze-dried connection part of self-healed G/H hydrogel, in which the dotted red line marks the adjacent between the G/H hydrogels.

Fig. 3

In vitro release curves and chondrogenic capacity of G + TGFβ1/sH-CD + KGN hydrogel.In vitro cumulative release curves of TGFβ1 (a) and KGN (b) in G/H, G/H-CD, G/sH, and G/sH-CD hydrogels after incubated in PBS (pH = 7.4) over 8 h to 14 days. (c) The expression levels of chondrogenic-related proteins (COL II and SOX9) in different groups via WB examination. (d) Immunofluorescence staining of COL II and SOX9 proteins for BMSCs-loaded hydrogels after 14 days in vitro culture. (e) COL II and GAG normalized to DNA in BMSCs-loaded hydrogels after 14 days in vitro culture. (f) Relative expression of chondrogenic-related genes (Col2a1 and Sox9) in BMSCs-loaded hydrogels after 14 days in vitro culture via qRT-PCR examination. *, p < 0.05; ns, no statistical significance.

Fig. 4

In vitro biocompatibility evaluation of G + TGFβ1/sH-CD + KGN hydrogel to BMSCs on day 1 and day 5. (a) Live/dead staining of BMSCs in control, G/sH-CD, G + TGFβ1/sH-CD, G/sH-CD + KGN, G + TGFβ1/sH-CD + KGN hydrogels. (b) F-actin/DAPI staining of BMSCs in control, G/sH-CD, G + TGFβ1/sH-CD, G/sH-CD + KGN, G + TGFβ1/sH-CD + KGN hydrogels. (c) Quantification analysis of OD value via the CCK-8 assay in various BMSCs-loaded hydrogels. (d) Quantitative analysis of DNA content in various BMSCs-loaded hydrogels. *, p < 0.05; ns, no statistical significance.

Fig. 5

In vivo evaluations of chondrogenesis and fused cartilage regeneration using BMSC-laden G + TGFβ1/sH-CD + KGN hydrogel in a rabbit. (a) Gross view, (b) H&E, Safranin-O, and immunohistochemical COL II stainings of BMSCs-loaded hydrogels with O-shape in various groups after 14 days of subcutaneous implantation. Quantification of (c) GAG content, (d) COL II content, and (e) Young's modulus for BMSCs-loaded hydrogels with O-shape in native tracheal cartilage ring and various hydrogel groups after 14 days subcutaneous implantation (*, p < 0.05; ns, no statistical significance). (f–g) Evaluation of the ability to regenerate fused cartilage using several self-healed O-shaped BMSCs-loaded G + TGFβ1/sH-CD + KGN hydrogels after 28 days of subcutaneous implantation, in which images in g are the magnified images of green dotted squares outlines zone in f. Black arrows marks the border between O-shaped BMSCs-loaded hydrogels. (h–i) Evaluation of the ability to regenerate fused cartilage using O-shaped BMSCs-loaded G + TGFβ1/sH-CD + KGN hydrogel adhered to native tracheal ring after 28 days of subcutaneous implantation, in which images in i are the magnified images of green dotted squares outlines zone in h. Black arrows marks the border between BMSCs-loaded hydrogel and native tracheal ring.

Fig. 6

Sutureless LSTD reconstruction using BMSCs-loaded G + TGFβ1/sH-CD + KGN hydrogel tracheal substitute in a rabbit. (a) Schematic illustration for LSTD reconstruction via sutured and unsutured procedures. (b) Photograph for BMSCs-loaded G + TGFβ1/sH-CD + KGN hydrogel tube for LSTD reconstruction with unsutured procedure. (c) Bronchoscope examination of tracheal lumen in sutured, unsutured, and native trachea groups, in which the red arrows denote the anastomosis. (d) Photograph for transplanted trachea in sutured and unsutured groups after 6 weeks LSTD reconstruction. Black arrows denote the anastomosis. (e) Survival rate of experimental rabbits in the sutured and unsutured groups 6 weeks after LSTD reconstruction. (f–g) Quantification of patency rate and sputum blockage in the sutured and unsutured groups (*, p < 0.05). (h–i) Overview of the transplanted trachea in sutured and unsutured groups with H&E and Safranin-O staining, shown in the left panels. Magnified views of the regions marked by black-dotted squares are shown with H&E, Safranin-O, and immunohistochemical COL II staining, as illustrated in the right panels. Similarly, magnified views of the green-dotted squares show immunofluorescence CK staining, also depicted in the right panels. Black dotted lines indicate the anastomosis, and black arrows point to granulation tissue hyperplasia. (j–l) Quantification of Young's modulus, COL II content, and epithelial thickness in sutured and unsutured groups (*, p < 0.05).